Endo-β-1,4-glucanase mutant and its gene, vector and preparation method

Abstract

The present invention discloses an endo-β-1,4-glucanase mutant and its gene, vector and preparation method. Including the following point mutations: mutating the 221st glutamic acid of the amino acid sequence shown in SEQ ID No.1 to valine or leucine, mutating the 117th lysine to methionine or valine; and / or mutating the 159th valine of the amino acid sequence shown in SEQ ID No.1 to lysine, asparagine or arginine. The present invention recombinantly constructs multiple endo-β-1,4-glucanase mutants with good thermal stability and pH tolerance by performing site-directed mutations on amino acids with similar spatial positions in adjacent β-folded structures of the endo-β-1,4-glucanase original enzyme from Thermobifida fusca. This feature effectively improves the availability of endo-β-1,4-glucanase under extremely harsh conditions in actual industrial production environments.

Description

Technical Field

[0001] The invention relates to an endoglucanase mutant and a gene, a vector and a preparation method thereof, and in particular to an endoglucanase mutant derived from Thermobifida fusca bacteria and a gene, a vector and a preparation method thereof. Background Art

[0002] Cellulose is a tough, fibrous, linear polymer of D-glucose units connected by β-1,4-glycosidic bonds. Cellulose is the most abundant polysaccharide in nature. As humanity looks to get rid of its dependence on fossil fuels and reduce its impact on the environment, cellulose has great economic value as a renewable bioenergy source. Cellulase can be produced by bacteria, fungi, plants and animals and is widely used in various industries, including food, pharmaceuticals, chemicals, textiles, agriculture and waste treatment. Cellulase is a group of glycosyl hydrolyzing complex enzymes that can effectively hydrolyze cellulose into fermentable sugars through the synergistic action of endoglucanases (EC3.2.1.4), exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). This method of using cellulase to treat and use cellulosic waste as a cheap carbon source is of great strategic significance.

[0003] The cellulases currently used in industrial production mainly come from Trichoderma reesei and Aspergillus oryzae. The cellulases produced by them are optimally PH 5.0-6.0 and the optimal temperature is between 50-70 degrees Celsius. When the enzyme solution is incubated in the temperature range of 70 to 90 degrees Celsius, the enzyme activity gradually decreases. When the temperature reaches 80 degrees Celsius, the enzyme activity decreases by about 30%. When the temperature reaches 90 degrees Celsius, the enzyme activity decreases by 80% or is completely inactivated. Due to the existence of high temperature scenes in actual industrial production environments, existing endoglucanases are difficult to maintain high catalytic activity in high temperature scenes, lack resistance to extremely short environments, resulting in a decrease in reaction rate, which is not conducive to increasing production capacity and reducing costs in the bioenergy industry. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an endo-β-1,4-glucanase mutant with high thermal stability and pH stability. The second purpose of the present invention is to provide a recombinant gene capable of expressing an endo-β-1,4-glucanase mutant. The third purpose of the present invention is to provide a vector capable of amplifying or expressing a mutant gene. The fourth purpose of the present invention is to propose a method for preparing an endo-β-1,4-glucanase mutant to solve the problem of how to transform the wild-type endo-β-1,4-glucanase by point mutation.

[0005] Technical solution: The endo-β-1,4-glucanase mutant described in the present invention comprises the following point mutation: glutamic acid at position 221 of the amino acid sequence shown in SEQ ID No.1 is mutated to valine or leucine.

[0006] Furthermore, the point mutation also includes: mutating the lysine at position 117 of the amino acid sequence shown in SEQ ID No. 1 to methionine or valine; and / or mutating the valine at position 159 of the amino acid sequence shown in SEQ ID No. 1 to lysine, asparagine or arginine.

[0007] Preferably, the amino acid sequence of the endo-β-1,4-glucanase mutant is any one of the sequences shown in SEQ ID No. 2-6, which are:

[0008] The glutamic acid at position 221 of the original enzyme sequence of endo-β-1,4-glucanase was replaced by valine;

[0009] The glutamic acid at position 221 of the original enzyme sequence of endo-β-1,4-glucanase was replaced by leucine and the lysine at position 117 was replaced by valine;

[0010] At the same time, the glutamic acid at position 221 of the original enzyme sequence of endo-β-1,4-glucanase was replaced by valine, the lysine at position 117 was replaced by methionine, and the valine at position 159 was replaced by asparagine;

[0011] The glutamic acid at position 221 of the original enzyme sequence of endo-β-1,4-glucanase was replaced by leucine and the valine at position 159 was replaced by lysine;

[0012] The 117th lysine of the original enzyme sequence of endo-β-1,4-glucanase was replaced by valine and the 159th valine was replaced by arginine.

[0013] In order to recombinantly express the above endo-β-1,4-glucanase mutant, the present invention provides a gene encoding the above endo-β-1,4-glucanase mutant protein.

[0014] The present invention artificially optimizes the sequence of the endo-β-1,4-glucanase gene (RCSB PDB code: 2CKS_B) from Thermobifida fusca, and its original amino acid sequence is shown in SEQ ID NO.1. The T.fusca CL enzyme has a total length of 307 amino acids and a theoretical molecular weight of 34.28 kDa. The present invention performs site-directed mutagenesis on amino acids that are close to adjacent β-folded structures in the secondary structure of the enzyme protein, namely, sites 117, 159, and 221. The position of the mutation site in the protein structure is shown in Figure 1 and 2 shown.

[0015] Preferably, the nucleotide sequence of the gene is shown as SEQ ID No.7-11.

[0016] Based on the gene sequence of the above endo-β-1,4-glucanase mutant protein, the present invention constructs a cloning vector or expression vector containing the above gene. The vector can be a plasmid or a virus.

[0017] In order to prepare the above-mentioned endo-β-1,4-glucanase mutant, the present invention provides a method for preparing an endo-β-1,4-glucanase mutant, comprising the following steps:

[0018] (1) Designing point mutation primers, using a plasmid carrying the wild-type endonuclease gene as a template, and using the point mutation primers to perform PCR reactions to obtain a mutant gene fragment containing bases and a linearized plasmid;

[0019] (2) connecting the mutant gene fragment to the linearized plasmid to construct an expression vector, and transferring the expression vector into the host bacteria for fermentation expression;

[0020] (3) Collecting host bacteria expressing the endo-β-1,4-glucanase mutant, resuspending the bacteria and breaking the cells, and centrifuging and collecting the supernatant to obtain a crude enzyme solution containing the endo-β-1,4-glucanase mutant.

[0021] Preferably, the point mutation primer sequence comprises:

[0022]

[0023]

[0024] . Primers E221V_F and CL_R were used to construct mutant gene fragments, and primers CL_F and E221V_R were used to construct linearized plasmids. Primers K117V_F and E221L_R were used to construct mutant gene fragments, and primers E221L_F and K117V_R were used to construct linearized plasmids. Primers K117M_F and V159N_R were used to construct mutant gene fragments, and primers V159N_F and E221V_R were used to construct mutant gene fragments, and primers E221V_F and K117M_R were used to construct linearized plasmids. Primers V159K_F and E221L_R were used to construct mutant gene fragments, and primers E221L_F and V159K_R were used to construct linearized plasmids. Primers K117V_F and V159R_R were used to construct mutant gene fragments, and primers V159R_F and K117V_R were used to construct linearized plasmids.

[0025] Preferably, the PCR reaction system is:

[0026]

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention recombinantly constructs multiple endo-β-1,4-glucanase mutants with good thermal stability and pH tolerance by performing site-directed mutations on the amino acids with similar spatial positions in the adjacent β-folding structure of the original endo-β-1,4-glucanase from Thermobifida fusca. The enzyme activity of the mutant enzyme at 95°C is 2.5 times higher than that of the original enzyme. At the same time, the enzyme activity is also significantly higher than that of the original enzyme in the pH range of 3.0-11.0, and the enzyme activity can be increased by up to 1.2 times. This characteristic effectively improves the availability of endo-β-1,4-glucanase under extremely harsh conditions in actual industrial production environments, has a wide range of applications, strong durability, and can be used on a large scale in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The protein structure of T.fusca CL enzyme and the amino acid positions 117 and 159;

[0029] Figure 2 The protein structure diagram of T.fusca CL enzyme and the amino acid position 221;

[0030] Figure 3 is the glucose standard curve;

[0031] Figure 4 is the enzyme activity of the original enzyme and mutant enzyme after heat treatment;

[0032] Figure 5 is the enzymatic activity of the original enzyme and the mutant enzyme under different pH conditions. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0034] Example 1: An endo-β-1,4-glucanase mutant E221V having a sequence as shown in SEQ ID No. 2, wherein the glutamic acid at position 221 of the original endo-β-1,4-glucanase sequence as shown in SEQ ID No. 1 is replaced by valine; the amino acid sequence of the original endo-β-1,4-glucanase is:

[0035] MTGTPVERYGKVQVCGTQLCDEHGNPVQLRGMSTHGIQWFDHCLTDSSLDALAYD

[0036] WKADIIRLSMYIQEDGYETNPRGFTDRMHQLIDMATARGLYVIVDWHILTPGDPHYN

[0037] LDRAKTFFAEIAQRHASKTNVLYEIANEPNGVSWASIKSYAEEVIPVIRQRDPDSVIIVG

[0038] TRGWSSLGVSEGSGPAEIAANPVNASNIMYAFHFYAASHRDNYLNALREASELFPVFV

[0039] TEFGTETYTGDGANDFQMADRYIDLMAERKIGWTKWNYSDDFRSGAVFQPGTCASGGPWSGSSLKASGQWVRSKLQS.

[0040] The nucleic acid sequence of the wild-type gene of endo-β-1,4-glucanase is:

[0041] ATGACCGGCACCCCGGTGGAACGCTATGGCAAAGTGCAAGTGTGCGGCACGCAGCTGTGCGATGAACATGGCAACCCGGTGCAGCTGCGCGGCATGAGCACGCATGGCATTCAGTGGTTTGATCATTGCCTGACCGATAGCAGCCTGGATGCGCTGGCGTATGATTGGAAAGCGGATATTATTCGCCTGAGCATGTATATTCAAGAAGATGGCTATGAAACCAACCCGCGCGGCTTTACCGATCGCATGCATCAGCTGATTGATATGGCGACCGCGCGCGGCCTGTATGTGATTGTGGATTGGCATATTCTGACCCCGGGCGATCCGCATTATAACCTGGATCGCGCGAAAACCTTTTTTGCGGAAATTGCGCAGCGCCATGCGAGCAAAACCAACGTGCTGTATGAAATTGCGAACGAACCGAACGGCGTGAGCTGGGCGAGCATTAAAAGCTATGCGGAAGAAGTGATTCCGGTGATTCGTCAGCGCGATCCGGATAGCGTGATTATTGTGGGCACCCGCGGCTGGAGTAGCCTGGGCGTGAGCGAAGGCAGCGGCCCGGCGGAAATTGCGGCCAACCCGGTGAACGCGAGCAACATTATGTATGCGTTTCATTTTTATGCGGCGAGCCATCGCGATAACTATCTGAACGCGCTGCGCGAAGCGAGCGAACTGTTTCCGGTGTTTGTGACCGAATTTGGCACCGAAACCTATACCGGCGATGGCGCGAACGATTTTCAGATGGCGGATCGCTATATTGATCTGATGGCGGAACGCAAAATTGGCTGGACCAAATGGAACTATAGCGATGATTTTCGCAGCGGCGCGGTGTTTCAGCCGGGCACCTGCGCGAGCGGTGGCCCGTGGAGCGGCAGTAGCCTGAAAGCGAGCGGTCAGTGGGTGCGCAGCAAACTGCAGAGC。

[0042] The preparation method of mutant E221V is as follows:

[0043] 1. Construction of recombinant plasmid

[0044] Plasmids pET-22b(+) and E. coli BL21(DE3) are both deposited by the applicant and are from commercial sources. The wild-type gene of endo-β-1,4-glucanase from Thermobifida fusca was synthesized by Jinweizhi (Suzhou) Co., Ltd. The site-directed mutagenesis sequence of the enzyme was constructed by the applicant using the PCR method. The primers used to introduce the mutation site are shown in Table 1. The template for the point mutation PCR is the pET-22b(+) plasmid with the wild-type gene sequence of endo-β-1,4-glucanase.

[0045] Table 1 Primers for mutant gene fragments and linearized plasmids

[0046]

[0047] Note: The underlined part in the primers is the mutation site, "F" represents the upstream primer, and "R" represents the downstream primer. Primers E221V_F and CL_R are used to construct the mutant gene fragment, and primers CL_F and E221V_R are used to construct the linearized plasmid.

[0048] Table 2 PCR reaction system

[0049]

[0050]

[0051] Table 3 PCR reaction conditions

[0052]

[0053] After the PCR product was verified by nucleic acid electrophoresis, it was purified using a DNA gel recovery kit. The reaction system was prepared as shown in Table 4, wherein the mutant gene fragment was a gene fragment in which glutamic acid at position 221 was replaced by valine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which glutamic acid at position 221 was replaced by valine. The reaction was carried out at 37°C for 1h for one-step cloning to construct an expression plasmid for the mutant enzyme. The gene sequence of the mutant enzyme is shown in SEQ ID No.7.

[0054] Table 4 One-step cloning system

[0055]

[0056] 2. Construction of recombinant strains and preparation of crude enzyme solution

[0057] The recombinant plasmid was transformed into E.coli DH5α competent cells, spread on the surface of LB solid culture medium containing 100μg / mL ampicillin, and cultured at 37℃ for 12h. All single colonies grown on the surface of the plate culture medium were scraped into a test tube containing 5mL LB liquid culture medium containing 100μg / mL ampicillin, and cultured at 37℃ and 200rmp for 12h. The plasmid was extracted with a plasmid extraction kit, and the recombinant plasmid was stored in a -20℃ refrigerator. The gene sequence of the mutant is shown in SEQ ID No.7.

[0058] The recombinant plasmid was transformed into the host bacteria E.coli BL21 (DE3), and spread on the surface of LB solid culture medium containing 100μg / mL ampicillin, and cultured at 37℃ for 8h. Pick a single E. coli colony and inoculate it into 3mL LB liquid culture medium containing 100μg / mL ampicillin, and culture it at 37℃ overnight as seed liquid. The seed liquid was inoculated at 5% and transferred to a 250mL conical flask containing 50mL TB culture medium, and cultured at 37℃ and 200rpm. The components of TB liquid culture medium are yeast powder 12g / L, tryptone 12g / L, glycerol 4ml / L, potassium dihydrogen phosphate 12.5g / L, potassium dihydrogen phosphate 2.3g / L. When the culture reaches 8h, add IPTG to a final concentration of 0.4mM, set the culture temperature to 22℃, and continue to culture for 24h. The fermentation broth was centrifuged, the bacteria were collected, resuspended in PBS buffer at pH 7.0, and the cells were disrupted by ultrasonic wave for 5-8 minutes until the liquid became slightly clear. The broth was centrifuged at 4000 g for 10 minutes, and the supernatant was used as the crude enzyme solution of mutant E221V.

[0059] Example 2: An endo-β-1,4-glucanase mutant K117V / E221L having a sequence as shown in SEQ ID No. 3, wherein the glutamic acid at position 221 of the original endo-β-1,4-glucanase sequence is replaced by leucine and the lysine at position 117 is replaced by valine.

[0060] The preparation method of mutant K117V / E221L is as follows:

[0061] The rest is the same as Example 1, except that the mutant gene fragment primer and the linearized plasmid primer are as follows:

[0062] Table 5 Primers for mutant gene fragments and linearized plasmid primers

[0063]

[0064] Primers K117V_F and E221L_R were used to construct mutant gene fragments, and primers E221L_F and K117V_R were used to construct linearized plasmids.

[0065] The PCR product was verified by gel electrophoresis and then recovered from the gel and stored at -20°C. The system was prepared according to Table 4, wherein the mutant gene fragment was a gene fragment in which the 117th lysine was replaced by valine and the 221st glutamic acid was replaced by leucine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which the 117th lysine was replaced by valine and the 221st glutamic acid was replaced by leucine. The expression plasmid of the mutant enzyme was constructed by incubating at 37°C for 1 hour. The gene sequence of the mutant enzyme is shown in SEQ ID No.8.

[0066] Example 3: An endo-β-1,4-glucanase mutant K117M / V159N / E221V having a sequence as shown in SEQ ID No. 4, wherein the glutamic acid at position 221 of the original endo-β-1,4-glucanase sequence is replaced by valine, the lysine at position 117 is replaced by methionine, and the valine at position 159 is replaced by asparagine.

[0067] The preparation method of mutant K117M / V159N / E221V is as follows:

[0068] The rest is the same as Example 1, except that the mutant gene fragment primer and the linearized plasmid primer are as follows:

[0069] Table 6 Primers for mutant gene fragments and linearized plasmid primers

[0070]

[0071]

[0072] Primers K117M_F and V159N_R were used to construct mutant gene fragment 1, primers V159N_F and E221V_R were used to construct mutant gene fragment 2, and primers E221V_F and K117M_R were used to construct linearized plasmid.

[0073] The PCR product was verified by gel electrophoresis and then recovered from the gel and stored at -20°C. The system was prepared according to Table 7, wherein the mutant gene fragment 1 was a gene fragment in which the 117th lysine was replaced by methionine and the 159th valine was replaced by asparagine, the mutant gene fragment 2 was a gene fragment in which the 159th valine was replaced by asparagine and the 221st glutamic acid was replaced by valine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which the 117th lysine was replaced by methionine and the 221st glutamic acid was replaced by valine. The expression plasmid of the mutant enzyme was constructed by incubating at 37°C for 2h.

[0074] Table 7 One-step cloning system

[0075]

[0076] The gene sequence of the mutant enzyme is shown in SEQ ID No.9.

[0077] Example 4: An endo-β-1,4-glucanase mutant V159K / E221L having a sequence as shown in SEQ ID No. 5, wherein the 221st glutamic acid in the original endo-β-1,4-glucanase sequence is replaced by leucine and the 159th valine is replaced by lysine.

[0078] The preparation method of the mutant V159K / E221L is as follows:

[0079] The rest is the same as Example 1, except that the mutant gene fragment primer and the linearized plasmid primer are as follows:

[0080] Table 8 Mutant gene fragment primers and linearized plasmid primers

[0081]

[0082] Primers V159K_F and E221L_R were used to construct mutant gene fragments, and primers E221L_F and V159K_R were used to construct linearized plasmids.

[0083] The PCR product was verified by gel electrophoresis and then recovered from the gel and stored at -20°C. The system was prepared according to Table 4, wherein the mutant gene fragment was a gene fragment in which the valine at position 159 was replaced by lysine and the glutamic acid at position 221 was replaced by leucine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which the valine at position 159 was replaced by lysine and the glutamic acid at position 221 was replaced by leucine. The expression plasmid of the mutant enzyme was constructed by incubating at 37°C for 1 hour.

[0084] The gene sequence of the mutant enzyme is shown in SEQ ID No.10.

[0085] Example 5: An endo-β-1,4-glucanase mutant K117V / V159R having a sequence as shown in SEQ ID No. 6, wherein the 117th lysine of the original endo-β-1,4-glucanase sequence is replaced by valine and the 159th valine is replaced by arginine.

[0086] The preparation method of mutant K117V / V159R is as follows:

[0087] The rest is the same as Example 1, except that the mutant gene fragment primer and the linearized plasmid primer are as follows:

[0088] Table 9 Mutant gene fragment primers and linearized plasmid primers

[0089]

[0090] Primers K117V_F and V159R_R were used to construct mutant gene fragments, and primers V159R_F and K117V_R were used to construct linearized plasmids.

[0091] The PCR product was verified by gel electrophoresis and then recovered from the gel and stored at -20°C. The system was prepared according to Table 4, wherein the mutant gene fragment was a gene fragment in which lysine at position 117 was replaced by valine and valine at position 159 was replaced by arginine, and the linearized plasmid was a linearized plasmid containing a gene fragment in which lysine at position 117 was replaced by valine and valine at position 159 was replaced by arginine. The expression plasmid of the mutant enzyme was constructed by incubating at 37°C for 1 hour.

[0092] The gene sequence of the mutant enzyme is shown in SEQ ID No.11.

[0093] Example 6: Determination of the enzyme activity of the endo-β-1,4-glucanase mutant enzymes prepared in Examples 1-5 at different temperatures

[0094] The enzyme activity was determined by the DNS method. Glucose solutions of 0.500, 0.375, 0.250, 0.188, and 0.125 mg / mL were prepared respectively. 150 μL of the solutions were taken into clean EP tubes. 150 μL of the DNS solutions were added respectively. The tubes were placed in a boiling water bath for 10 min. The absorbance was measured at 540 nm and a standard curve was drawn. Figure 3 As shown. Add 50μL crude enzyme solution and 100μL 1% sodium carboxymethyl cellulose solution to the EP tube, react at 55℃ for 20min, quickly cool to room temperature, add 150μL DNS solution to stop the reaction, and boil in a boiling water bath for 10min. After waiting for cooling, dilute appropriately with deionized water and measure the absorbance at 540nm. In addition, take an equal amount of crude enzyme solution and add 100μL deionized water, use the same DNS solution and treatment method as a negative control. The enzyme activity is calculated according to the above method. 1 enzyme activity is defined as the amount of enzyme that catalyzes the decomposition of 1μmol of reducing sugar in 1 minute under the set reaction conditions.

[0095] The crude enzyme solutions extracted in Examples 1-5 were incubated at 55°C, 65°C, 75°C, 85°C, and 95°C for 3 hours, and then catalytic reaction was carried out at 65°C and enzyme activity was measured to screen mutants with improved enzyme activity compared to the original enzyme. The results are as follows: Figure 4 The mutants of the enzyme still have high enzyme activity after high temperature treatment. After incubation at 85℃ for 3 hours, the enzyme activity of mutants E221V, K117V / E221L, and K117M / V159N / E221V increased by more than 21% compared with the original enzyme; after incubation at 95℃ for 3 hours, the enzyme activity of mutants E221V and K117M / V159N / E221V increased by 2.5 times compared with the original enzyme.

[0096] Example 7: Determination of the enzyme activity of the endo-β-1,4-glucanase mutant enzymes prepared in Examples 1-5 under different pH conditions

[0097] The recombinant bacteria induced by IPTG and cultured for 24 h in Examples 1-5 were centrifuged and collected, and the cells were resuspended in 1 ml of pH 3.0, pH 5.0 sodium hydrogen phosphate-citrate buffer, pH 7.0, pH 9.0, and pH 11.0 phosphate buffer. The remaining steps were carried out as in Example 2 to prepare the crude enzyme solution, and the enzyme activity was determined as in Example 3. The mutants with improved enzyme activity compared to the original enzyme were screened, and the results were as follows: Figure 5 As shown. Under acidic conditions of pH 3.0 and pH 5.0, the enzyme activity of mutant K117V / V159R was increased by 50% and 104% compared with the original enzyme. Under alkaline conditions of pH 9.0 and pH 11.0, the enzyme activity of mutants E221V and V159K / E221L was increased compared with the original enzyme. Among them, at pH 9.0, the enzyme activity of V159K / E221L was increased by 49% compared with the original enzyme. At pH 11.0, the enzyme activity of E221V was increased by 1.2 times compared with the original enzyme.

Claims

1. An endo-β-1,4-glucanase mutant, characterized in that: The mutant is: the glutamic acid at position 221 of the amino acid sequence shown in SEQ ID No.1 is mutated to valine, or the lysine at position 117 is mutated to valine and the glutamic acid at position 221 is mutated to leucine, or the lysine at position 117 is mutated to methionine and the valine at position 159 is mutated to asparagine and the glutamic acid at position 221 is mutated to valine, or the valine at position 159 is mutated to lysine and the glutamic acid at position 221 is mutated to leucine, or the lysine at position 117 is mutated to valine and the valine at position 159 is mutated to arginine; the amino acid sequence of the mutant is any one of the sequences shown in SEQ ID No.2-6.

2. A gene encoding the endo-β-1,4-glucanase mutant protein according to claim 1.

3. The gene encoding the endo-β-1,4-glucanase mutant protein according to claim 2, characterized in that: The nucleotide sequence of the gene is any one of the sequences shown in SEQ ID No.7-11.

4. A cloning vector or expression vector containing the gene according to claim 2.

5. The method for preparing the endo-β-1,4-glucanase mutant according to claim 1, characterized in that: The steps include: (1) Designing point mutation primers and linearization primers, using the plasmid carrying the wild-type endonuclease gene as a template, and performing PCR reactions with the point mutation primers and linearization primers to obtain the mutant gene fragment and linearized plasmid respectively; (2) Connecting the mutant gene fragment to the linearized plasmid to construct an expression vector, and then transferring the expression vector into the host bacteria for fermentation expression; (3) Collect the host bacteria expressing the endo-β-1,4-glucanase mutant, resuspend the bacteria, break the cells, and centrifuge to obtain the supernatant to obtain the crude enzyme solution containing the endo-β-1,4-glucanase mutant.

6. The method for preparing the endo-β-1,4-glucanase mutant according to claim 5, characterized in that: The annealing temperature of the PCR reaction is 55-60°C.

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